1. Field of the Invention
The invention relates to a method for determining the local distribution of a quantity to be measured relative to or present in a predefined measurement area of a biological sample, preferably the surface of an organ or the epidermis, where in a first measuring process for determining a first measurement variable a sensor film with a luminescence indicator reacting to this variable by a change of at least one optical characteristic is applied to the measurement area and the first measurement variable is detected by imaging means, as well as to a system for implementation of this method.
2. The Prior Art
For a variety of medical applications, especially in the diagnostic sector, it is of prime importance that the local distribution of a measurement variable over the surface of an organ, such as the human skin, or the distribution of the flow rate of a given substance through an interface, should be determined. Besides, such measured results are useful in checking and controlling methods of medical therapy.
Tissue oxygenation, for example, is an important parameter in diagnosing diseases resulting from disturbed microcirculation. Oxygen supply is determined on the one hand by the perfusion properties of the blood, and on the other hand by transcutaneous transport properties and oxygen consumption in the skin. In addition, oxygen supply is partly effected by oxygen absorption from the environment, an activity known as cutaneous respiration. Problems occur when several parameters influencing the medically relevant variable to different degrees, are to be detected simultaneously.
Determining oxygen concentration or transcutaneous oxygen partial pressure (tcPO2) by means of skin electrodes is a technique well known in the art, as is the use of optical sensors based on fluorescence quenching. The latter do not consume oxygen during measuring, which is an advantage over the use of electrodes.
Another known technique is the so-called FLIM (fluorescence lifetime imaging) process using optical sensors on the basis of fluorescence quenching, where a sensor membrane carrying a suitable luminescence indicator is attached to the skin region to be examined. Oxygen diffused through the skin surface enters the sensor membrane, and fluorescence quenching resulting therefrom may be analyzed by the detector system.
In this context apparatus and method for measuring tissue oxygenation are described in U.S. Pat. No. 5,593,899, where oxygen-dependent quenching of a fluorescence indicator is used for measurement. The oxygen supply of the skin is determined by applying a luminescent probe within a skin cream to a suitable area of the skin, and covering that area by an oxygen-impermeable film. The optical means for excitation of the indicator and detection of the respective radiation is encased in a housing whose transparent cover is directly placed over the O2-impermeable film. An interference filter and a photodiode are added to this set-up. The luminescent probe is subject to excitation radiation from a modulated radiation source via optical fiber guides. The above set-up is suitable only for integral measuring over the entire area covered by the optical equipment. It is impossible with this system, however, to obtain accurate information on boundary regions between sufficiently oxygenated and inadequately oxygenated skin regions.
The device disclosed in EP 0 516 610 B1 can be used for measuring not only oxygen concentration, but also oxygen flow through an interface, for example, a skin area. The sensor layer of the device to be associated with the interface offers a known, finite resistance to the oxygen flow to be measured, and is provided with at least one optical indicator determining oxygen concentration on one side of the sensor layer. From the concentration value measured on one side of the sensor layer and known beforehand on the other side (ambient air) the material flow through the interface is determined. According to a variant of the invention the sensor layer may be scanned a really by means of an imaging system (CCD), so that local distribution of oxygen flow or oxygen concentration may be measured.
Further ideas and measured results regarding local distribution of oxygen flow and subcutaneous oxygen concentration, as well as a proposal for a measuring process by imaging means are disclosed in the paper “Fluorescence Lifetime Imaging of the Skin PO2: Instrumentation and Results” in Advances in Experimental Medicine and Biology, Vol. 428, pp 605–611 (1997), published by Plenum Press N.Y. The paper describes a sensor membrane for measuring transcutaneous oxygen concentration, which comprises an optical insulating layer facing the skin surface, a sensing layer with a luminescence indicator with O2-sensitive decay time, and a supporting layer that is impermeable to oxygen. Further described is a membrane for measuring oxygen flow, which differs from the above sensor membrane by featuring a diffusion barrier with known oxygen permeability instead of the O2-impermeable layer. For the purpose of measurement the sensor membrane is applied to the measuring surface, for example, a skin area. The measuring process employs a modulation technique, where the LEDs emitting excitation radiation in the direction of the sensor membrane are actuated by a square-wave generator and emit square-wave modulated excitation radiation. The emission radiation emitted by the sensor membrane is detected by a CCD camera with modulated amplification and passed on pixel by pixel to a computing unit for image-processing. The oxygen distribution measured in a polymeric layer is documented as an image of the variations in oxygen distribution over an area of the skin.
Other imaging processes for measurement by means of phase fluorometry are described in U.S. Pat. No. 5,485,530.
A complete estimate of the oxygen supply of a certain area of the skin or surface of an organ can only be made if the oxygen status of the inspected region is complemented by information on blood supply and perfusion rate.
A number of well-advanced methods and devices are at disposal for obtaining the necessary information on perfusion, such as Laser-Doppler flow measurements (see U.S. Pat. No. 4,476,875), by means of which local perfusion of the blood vessels may be examined on the basis of the frequency shift of radiation emitted by a laser lightsource. It would also be possible to expand emission radiation by suitable optical means, or measurement areas could be scanned step by step with the use of a laser beam, so that a picture will be obtained of the local distribution of the perfusion rate. Such methods have become known as Laser-Doppler imaging processes (LDI). The result of a Laser-Doppler measurement will depend on the velocity and number of red blood cells scattering the laser light.
In U.S. Pat. No. 4,862,894, for example, a system for analyzing the bloodstream in an area of the skin is described, where a laser beam is used which is expanded by a cylindrical lens. In one variant the skin surface is scanned by the laser line by line, and a two-dimensional image is detected of how the flow velocity of the blood is distributed. Another method and apparatus for measuring fluids in motion is described in U.S. Pat. No. 5,361,769, where the area of a specimen is scanned by a Laser-Doppler imaging process. Via a lens combination in the laser beam varying object distances are compensated, thus increasing measuring accuracy.
So far it has not been possible to obtain satisfactory measured results on the oxygen supply of an organ or a skin area as the quantities to be measured usually vary at a rate that is faster than the rate at which the different measuring devices or processes required therefor can be successively employed in the skin area to be analyzed. A further problem is presented by the heterogeneity of the area to be measured, e.g., the surface of the skin, so that point measurements such as electrode or Laser-Doppler flow measurements are not successful whilst imaging systems and processes such as FLIM and LDI can only be used in adjacent sites or one after the other. For example, when tests on one and the same skin area switch from an FLIM to LDI system, this will take too long for parameters like perfusion and oxygen status, which frequently change within seconds, thus preventing meaningful measurement. When devices have to be exchanged, it will be difficult to reposition them precisely, so that measured areas are likely to vary slightly.
Using previous measuring systems as a basis, it is the object of this invention to propose a method and apparatus for determining local distribution of several measurement variables in a predefined measurement area of a biological sample, which should enable the user to obtain information on physiological parameters and their localization.